I. We have characterized the glycosylation sites of human beta- hexosaminidase B and have determined the effect of individual oligosaccharides on the catalytic activity, transport and processing of the enzyme. Glycosylation is an essential step for the ultimate expression of lysosomal enzymes because it is only after the construction of a mannose-6-phosphate recognition marker that the enzymes are recognized by a receptor and delivered to lysosomes. The five potential glycosylation sites (Asn-X-Ser/Thr) of the hexosaminidase beta-chain were individually modified by site-directed mutagenesis and the constructs were expressed in Cos 1 cells under control of the SV-40 late promotor. By this analysis, we determined that four of the five potential glycosylation sites were modified by addition of oligosaccharide chains. The lack of any one of the oligosaccharides did not dramatically affect catalytic activity or the delivery of the enzyme to lysosomes. We also demonstrated a selectivity in the phosphorylation of the oligosaccharides on hexosaminidase B; two of the four oligosaccharide chains were predominantly phosphorylated. When these two phosphorylated sites were removed by mutagenesis, the enzyme was not delivered to lysosomes. This work has also clarified the peptide structure of the mature enzyme. II. A hexosaminidase alpha chain cDNA has been cloned from a library prepared with the fibroblast mRNA of a patient with the adult form of Tay-Sachs disease. We are currently sequencing the clone to identify the mutation responsible for this form of the disease.